8-11h_E3_Rep1
Protocols: Freshly hatched adults of the appropriate genotype were placed in embryo collection vials with standard apple cap plates. Drosophila embryos were collected on apple juice agar plates at 25 °C at the appropriate time-point (after 3 pre-lays of 45 minutes for stage-specific collections), dechorionated using 50% bleach, and washed alternately with water and PBS + 0.1% Triton X-100. The embryos were covalently crosslinked in 1.8% formaldehyde for 15 min at room temperature, quenched with 2M Tris-HCl pH7.5 for 5 min, washed in cold PBS + 0.1% Triton X-100, and stored at -80°C.  About 5000 frozen embryos were homogenized in 10 ml of chilled cell lysis buffer (10mM Tris-HCl, pH8; 10 mM NaCl; 0.2% IGEPAL + Complete) on ice. The lysate was incubated 15 min on ice, and spun at 3,000g for 5 min to pellet the nuclei. The nuclei were then washed in cell lysis buffer, spun at 3,000g for 5 min and resuspended in 50 µl of 0.3% SDS before 10 min incubation at 37C. 148µl of water and 25µl of 10% Triton X-100 were added to the nuclei before incubating for an additional 15 min at 37C. 25µl of 10x CutSmart buffer and 25U of the MboI restriction enzyme were added, and the nuclei were incubated for 90 min at 37C, shaking gently. Another 25U of the MboI were added and the nuclei incubated for another 90 min.  The nuclei were then incubated for 20 min at 62C to inactivate the enzyme. The following components were added before incubating for 2 hours at 25C with gently shaking: 556µl of water, 100µl of 10x ligation buffer, 80µ of 10% Triton, 12µl of 10mg/ml BSA, and 2000U of T4 DNA ligase. Another 2000U of T4 DNA ligase was added and the nuclei incubated for another 2 hours. The nuclei were spun at 2500g for 5min and the pellet ressuspended in elution buffer (10mM Tris-HCl, pH8; 1 mM EDTA; 0.5M NaCl; 1% SDS solution; 2µg/µl proteinase K) and incubated 30 min at 55C then overnight at 65C. The next day, 100μg of RNaseA were added to the reaction before 15min incubation at 37C. The DNA was purified using the Zymo ChIP DNA Clean & Concentrator kit and elute in 50 µL of 10 mM Tris-Cl pH 8.0. The DNA was then digested with NlaIII overnight at 37C. The next day, the sample was incubated for 20 min at 65C to inactivate the enzyme. The second liation was carried out using 200U of T4 DNA ligase for 4 hours at 16C then 30 min at room temperature. The sample was purified using Nucleomag PCR beads and eluted in 300µl of 10 mM Tris-HCl pH 8.0. The DNA was amplified from 320 ng of 4C template for 27 cycles using the Q5 Hot Start DNA Polymerase. The PCR product was purified using SPRIselect beads (Beckman Coulter) and 100 ng of each PCR product was used to generate the final libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). The libraries were indexed for multiplexing using NEBNext multiplex oligos kit for Illumina (New England Biolabs).
strain
wild type
viewpoint
E3
individual
mixed pool of embryos
organism
Drosophila melanogaster
organism
Drosophila melanogaster
collection date
not collected
sex
mixed sex
scientific_name
Drosophila melanogaster
common name
fruit fly
organism part
whole embryo
developmental stage
embryonic stage 12-14
genotype
y[1] w[1118]
isolate
not applicable
age
8-11
geographic location (country and/or sea)
not collected